Thyrotropin or thyroid stimulating hormone (TSH) is a pituitary hormone that regulates thyroid function via the TSHR (Szkudlinski M W, Fremont V, Ronin C, Weintraub 2002 Thyroid-stimulating hormone and TSHR structure-function relationships. Physiological Reviews 82: 473-502). Binding of TSH to the TSHR triggers receptor signaling which leads to stimulation of formation and release of thyroid hormones; thyroxine (T4) and tri-iodothyronine (T3). A feedback mechanism involving the levels of T4 and T3 in the circulation and thyrotropin releasing hormone (TRH) secreted by the hypothalamus controls the release of TSH that in turn controls thyroid stimulation and the levels of thyroid hormones in serum.
The TSHR is a G-protein coupled receptor and has three domains: a leucine rich domain (LRD), a cleavage domain (CD) and a transmembrane domain (TMD) (Núñez Miguel R, Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Blundell T L, Rees Smith B, Furmaniak J 2004 Analysis of the thyrotropin receptor-thyrotropin interaction by comparative modeling. Thyroid 14: 991-1011). The TSHR shows amino acid and structural similarities with the other glycoprotein hormone receptors ie luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). The structure of the FSHR in complex with its ligand (ie FSH) has been solved at 2.9 Å resolution (Fan Q R, Hendrickson W A 2005 Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433: 269-277).
It is well documented in the art that some patients with autoimmune thyroid disease (AITD), which is the most common autoimmune disease affecting different populations worldwide, have autoantibodies reactive with the TSHR (Rees Smith B, McLachlan S M, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocrine Reviews 9: 106-121). In the majority of cases, these autoantibodies bind to the TSHR and mimic the actions of TSH thereby stimulating the thyroid to produce high levels of T4 and T3. These autoantibodies are described as thyroid stimulating autoantibodies, or TSHR autoantibodies (TRAbs) with stimulating activity or TSH agonist activity. The feedback mechanism which usually controls thyroid function is no longer effective in the presence of thyroid stimulating autoantibodies and the patients present with symptoms of a hyperactive thyroid (excess of thyroid hormones in serum). This condition is known as thyrotoxicosis or Graves' disease. In some patients the TRAbs with stimulating activity are thought to interact with TSHRs in retro-orbital tissues and contribute to causing the eye signs of Graves' disease.
In some patients with AITD, autoantibodies bind to the TSHR, preventing TSH from binding to the receptor but have no ability to stimulate TSHR activity; these types of autoantibodies are known as TRAbs with blocking activity or TSH antagonist, activity.
TSHR autoantibodies when present in serum of pregnant women in high concentrations can cross the placenta and may cause neonatal thyrotroxicosis (in the case of stimulating autoantibodies) or neonatal hypothyroidism (in the case of blocking autoantibodies) (Rees Smith B, McLachlan S M, Furmaniak J 1988 supra).
A human monoclonal autoantibody which acts as a powerful thyroid stimulator (hMAb TSHR1) has been described in detail in International Patent Application WO2004/050708A2. The binding site for hMAb TSHR1 has been found to be located on the surface of the TSHR leucine rich domain (LRD) and overlaps extensively with the binding site for TSH. However, the binding pocket for TSH or hMAb TSHR1 is conformational and involves discontinuous regions of the TSHR folding together. Characterization of the binding site for hMAb TSHR1 in detail, in particular the important contact amino acids in the interaction between the TSHR and hMAb TSHR1, is of critical importance in studies which aim to improve the diagnosis and management of diseases associated with an autoimmune response to the TSHR.
International patent application WO 2006/016121A discloses a mutated TSHR preparation including at least one point mutation which can be used in the differential screening and identification of patient serum stimulating TSHR autoantibodies, patient serum blocking TSHR autoantibodies and thyroid stimulating hormone in a sample of body fluid from a patient which is being screened. The invention described in international patent application number WO2006/016121A provides useful information regarding the regions of the TSHR which are important in the interaction with various antibodies including hMAb TSHR 1, a mouse monoclonal antibody (9D33) with TSHR blocking activity and with TSH. However, details of the interactions between amino acids in the TSHR and amino acids in hMAb TSHR1 at the atomic level could not be derived from even the best experimental studies, such as those described in WO2006/016121A, which involved mutating the TSHR.
The present invention is based on the preparation of a complex formed by a fragment of the TSHR LRD (which is involved in forming the binding pocket for TSH and hMAb TSHR1) and the Fab fragment of hMAb TSHR1. The hMAb TSHR1 preparations described in this specification are referred to as M22 for convenience. M22 IgG can be purchased from RSR Ltd. The TSHR fragment covering amino acids 1-260 (TSHR260) in complex with hMAb TSHR1 (M22) Fab fragment is referred to as TSHR260-M22. A TSHR260-M22 complex was purified, concentrated and crystallized. The data from X-ray diffraction were used to solve the structure of TSHR260 as described in the present invention. The structure of M22 Fab solved at 1.65 Å resolution has been described before (Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana L D K E, Chirgadze D Y, Núñez Miguel R, Blundell T L, Furmaniak J, Rees Smith B 2004 Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function. Thyroid 14: 560-570). The structure of M22 in the complex was compared to that of un-bound M22. The interactions between TSHR260 and M22 were then refined at the atomic level.
To date highly purified TSHR preparations with their TSH and TRAb binding activity intact have not been available. The purified TSHR preparations described in the art were denatured in part and not pure or homogenous enough for crystallisation.